Production and characterization of two monoclonal antibodies specific for Plasmopara halstedii

Sunflower downy mildew, caused by the fungus Plasmopara halstedii, is a potentially devastating disease. We produced two monoclonal antibodies (MAbs) (12C9 and 18E2) by immunizing mice with a partially purified extract of P. halstedii race 1. Both MAbs detected in enzyme-linked immunosorbent assay (ELISA) all races of P. halstedii present in France. No cross-reactions were observed with Plasmopara viticola or with other fungi commonly associated with sunflowers. Both MAbs recognized the same three fungal antigens with molecular masses of 68, 140, and 192 kDa. However, the epitopes on the fungal antigens were distinct and repetitive. Seed homogenates from infected plants were incubated in wells coated with MAb 18E2. This resulted in the trapping of P. halstedii antigens that were identified with biotinylated MAb 12C9. No reactions were seen with seed homogenates from healthy plants. Thus, our results suggest that these MAbs might be used to develop a sandwich ELISA detection system for P. halstedii in infected seeds.     

A Method to Measure Aggressiveness of Plasmopara halstedii (Sunflower Downy Mildew)

For the first time, a method was used to measure aggressiveness of two Plasmopara halstedii races (100 and 710), the parasite causing sunflower downy mildew. Two sunflower lines showing different levels of quantitative resistance were used to measure two aggressiveness criteria: latent period and sporulation density. A strain of race 100 had a shorter latent period and greater sporulation density than a strain of race 710. The sunflower inbred line BT, rather susceptible in the field, presented a greater sporulation density and a shorter latent period than another inbred line FU, which shows greater resistance in the field. These results indicated that race 100 was more aggressive than race 710. The behaviour in the field of the two inbred lines was confirmed in the laboratory observations.     

Cloning,sequence and characterization of a sunflower (Helianthus annuus L.) pathogen-induced gene showing sequence homology with auxin-induced genes from plants

The establishment of a plant-pathogen interaction involves changes in gene expressions in both organisms. To isolate Helianthus annuus genes whose expression is induced during processes of resistance to Plasmopara halstedii, a comparison of the expression pattern of healthy sunflowers was made with sunflowers infected with 2 races of P. halstedii, either virulent or avirulent, using differential display of mRNA. A full-length cDNA, HaAC1, representing a sunflower gene whose expression is enhanced during early stages of the incompatible interaction, was isolated. Different timing of RNA accumulation is observed between compatible and incompatible combinations. Sequence analysis and database search revealed significant homology with auxin-induced genes from plants. The expression of this gene, is also induced after treatment with 2,4-dichlorophenoxyacetic acid (2,4-D), salicylic acid (SA) and wounding.     

Emerging virulence arising from hybridisation facilitated by multiple introductions of the sunflower downy mildew pathogen Plasmopara halstedii

The sunflower downy mildew pathogen Plasmopara halstedii is an invasive plant pathogen in Europe of American origin. Despite efforts to produce resistant host varieties, nationwide monitoring in France has revealed the rapid emergence of new virulent races increasing the number from one founder identified in 1966 to as many as 14 today. We have genotyped 146 samples (including all 14 races) using 13 nuclear and one mtDNA marker. Samples of the same race were found to share alleles/mtDNA haplotype and the two most common races had individuals with multiple matching genotypes. Cluster analyses confirmed that the samples form three groups to which races strongly adhere. Clusters were highly differentiated (F(ST) 0.65) and characterised by high inbreeding coefficients. Despite this, samples of recently emergent races, including six that are unique to France had mixed ancestry between the groups suggesting they have arisen in situ due to hybridisation. Five such samples also had conflicting mtDNA and nuclear DNA profiles. This demonstrates that multiple introductions have aided the establishment of this pathogen in France, and suggests recombination facilitated by these introductions is driving the emergence of new and endemic races in response to host resistance.     

Effect of Host-race Combination on Resistance of Sunflower, Helianthus annuus L., to Downy Mildew Plasmopara balstedii

Study of resistance of sunflower, Helianthus annuus L., to downy mildew, Plasmopara halstedii , shows that both susceptible and resistant seedlings are always infected by the fungus, then hypersensitive-like reactions occur in the parenchyma and necroses form around the parasite. Comparison of 21 incompatible combinations (resistance) shows that the extent of the fungus growth differs according to the host-race combination. With type I resistance, the fungus is limited to the roots andthe lower part of the hypocotyl. With the type II resistance, the fungus grows throughout the whole length of the hypocotyl and may sporulate on the cotyledons.     

Resistance of sunflower to the biotrophic oomycete Plasmopara halstedii is associated with a delayed hypersensitive response within the hypocotyls

The biotrophic oomycete Plasmopara halstedii is the causal agent of downy mildew in sunflower. It penetrates the roots of both susceptible and resistant sunflower lines and grows through the hypocotyls towards the upper part of the seedling. RT-PCR analysis has shown that resistance is associated with the activation of a hsr203J-like gene, which is a molecular marker of the hypersensitive reaction in tobacco. Activation of this gene was specifically observed during the incompatible interaction and coincided with cell collapse in the hypocotyls. This HR was also associated with the early and local activation of the NPR1 gene which is a key component in the establishment of the SAR. No such HR or a significant activation of the hsr203J-like gene were observed during the compatible combination. These results suggest that the resistance of sunflower to P. halstedii is associated with an HR which fails to halt the parasite. By contrast, this HR triggers a SAR which takes places in the upper part of the hypocotyls and eventually leads to the arrest of parasite growth. A model describing the resistance of plants to root-infecting oomycetes is proposed.     

Transcripts of sunflower antioxidant scavengers of the SOD and GPX families accumulate differentially in response to downy mildew infection, phytohormones, reactive oxygen species, nitric oxide, protein kinase and phosphatase inhibitors

Messenger RNA accumulation of two previously characterized sunflower glutathione peroxidases (GPXha-1 and GPXha-2) was monitored in response to pathogen attack. The accumulation of GPXha-1 and GPXha-2 mRNAs was also followed after stimulation with various signalling molecules including stress related phytohormones, reactive oxygen species, nitric oxide and protein phosphatase or kinase inhibitors. To have a more complete view of the response of the plant enzymatic antioxidant system when challenged by these various stimuli, the accumulation of superoxide dismutase (SOD) mRNAs was monitored too. To do so, partial sunflower SOD cDNAs (SODha-1 and SODha-2) were cloned in the course of the study. We show here that sunflower GPX mRNA accumulated differently in leaves of plants infected with either a virulent or an avirulent race of pathogen ( Plasmopara halstedii ). We also observed that any of the stimuli used translated in a stronger accumulation of both GPX mRNAs. These data suggest that GPX enzymes are involved in the hypersensitive and stress responses in sunflower. When compared to each other, GPX and SOD messenger steady state levels behaved differently following biotic stress or treatments with stress signalling factors. This suggests that the antioxidant enzymes GPX and SOD are likely to play different functions in stress responses.     

Histopathological Studies of Resistance of Sunflower (Helianthus annuus L.) to Downy Mildew (Plasmopara halstedii)

Histopathological studies of the infection of sunflower seedlings by downy mildew ( Plasmopara halstedii ) have shown that penetration of roots and the lower part of the hypocotyl occurs for both compatible combinations (suseptibility) and incompatible combinations (resistance). After penetrating susceptible genotypes, the parasite develops intercellular hyphae and intracellular haustoria, leading to systemic invasion. In contrast, in resistant plants, as soon as colonization develops, hypersensitive-like reactions occur in the parenchyma, with the appearance of necrotic zones surrounded by dividing cells. Growth of the parasite is strongly inhibited and most hyphae are blocked before they reach the cotyledonary node.     

A RAPD Assay for Strain Typing of the Biotrophic Grape Powdery Mildew Fungus Uncinula necator Using DNA Extracted from the Mycelium

Délye, C., Corio-Costet, M.-F., and Laigret, F. 1995. A RAPD assay for strain typing of the biotrophic grape powdery mildew fungus Uncinula necator using DNA extracted from the mycelium. Experimental Mycology 19, 234-237. We describe, for the first time, a RAPD assay using DNA extracted from the mycelium of a powdery mildew fungus, Uncinula necator, a pathogen of grape. No contamination by plant DNA was observed, and the resulting patterns were fully repetitive. RAPD profiles were unchanged when using two different DNA polymerases or three different thermocyclers. Thirteen strains were tested for amplification, using 95 primers. Only 4% of the amplified fragments were polymorphic. Cluster analysis revealed that the strains from the same geographical origin had the higher genetic similarity, suggesting a short-range dissemination of U. necator. This RAPD assay was also successfully applied to the grape downy mildew fungus, Plasmopara viticola, indicating that it can be used for other fungi which cannot be grown on artificial media.     

Identification of non-TIR-NBS-LRR markers linked to the <Emphasis Type="Italic">Pl5</Emphasis>/<Emphasis Type="Italic">Pl8</Emphasis> locus for resistance to downy mildew in sunflower

The resistance of sunflower, Helianthus annuus L., to downy mildew, caused by Plasmopara halstedii, is conferred by major genes denoted by Pl. Using degenerate and specific primers, 16 different resistance gene analogs (RGAs) have been cloned and sequenced. Sequence comparison and Southern-blot analysis distinguished six classes of RGA. Two of these classes correspond to TIR-NBS-LRR sequences while the remaining four classes correspond to the non-TIR-NBS-LRR type of resistance genes. The genetic mapping of these RGAs on two segregating F2 populations showed that the non-TIR-NBS-LRR RGAs are clustered and linked to the Pl5/ Pl8 locus for resistance to downy mildew in sunflower. These and other results indicate that different Pl loci conferring resistance to the same pathogen races may contain different sequences.     

Molecular variability of sunflower downy mildew, Plasmopara halstedii, from different continents

Downy mildew of sunflower (Helianthus annuus L.), caused by the pathogen Plasmopara halstedii, is a potentially devastating disease. Seventy-seven isolates of P. halstedii collected in twelve countries from four continents were investigated for RAPD polymorphism with 21 primers. The study led to a binary matrix, which was subjected to various complementary analyses. This is the first report on the international genetic diversity of the pathogen. Similarity indices ranged from 89% to 100%. Neither a consensus unweighted pair group method with arithmetic means (UPGMA) tree constructed after bootstrap resampling of markers nor a principal component analysis based on distance matrix revealed very consistent clusterings of the isolates, and groups did not fit race or geographical origins. Phylogenies were probably obscured by limited diversity. Analysis of molecular variance (AMOVA) and Nei's genetic diversity statistics gave similar conclusions. Most of the genetic diversity was attributable to individual differences. The most differentiated races also had the lowest within-diversity indices, which suggest that they appeared recently with strong bottleneck effects. Our analyses suggest that this pathogen is probably homothallic or has an asexual mode of reproduction and that gene flow among countries can occur through commercial exchanges. Knowledge of the downy mildew populations' structure at the international level will help to devise strategies for controlling this potentially devastating disease.     

A simple and rapid method for detection of Trypanosoma evansi in the dromedary camel using a nested polymerase chain reaction

A nested polymerase chain reaction (nPCR)-based assay, was developed and evaluated for rapid detection of Trypanosoma evansi in experimentally infected mice and naturally infected camels (Camelus dromedarius). Four oligonucleotide primers (TE1, TE2, TE3 and TE4), selected from nuclear repetitive gene of T. evansi, were designed and used for PCR amplifications. The first amplification, using a pair of outer primers TE1 and TE2, produced a 821-bp primary PCR product from T. evansi DNA. The second amplification, using nested (internal) pair of primers TE3 and TE4, produced a 270-bp PCR product. T. evansi DNAs extracted from blood samples of experimentally infected mice and naturally infected Sudanese breed of dromedary camels were detected by this nested PCR-based assay. The nested primers TE3 and TE4 increased the sensitivity of the PCR assay and as little as 10 fg of T. evansi DNA (equivalent to a single copy of the putative gene of the parasite) was amplified and visualized onto ethidium bromide-stained agarose gels.     

Candidate disease resistance genes in sunflower cloned using conserved nucleotide-binding site motifs: genetic mapping and linkage to the downy mildew resistance gene Pl1.

Disease resistance gene candidates (RGCs) belonging to the nucleotide-binding site (NBS) superfamily have been cloned from numerous crop plants using highly conserved DNA sequence motifs. The aims of this research were to (i) isolate genomic DNA clones for RGCs in cultivated sunflower (Helianthus annuus L.) and (ii) map RGC markers and Pl1, a gene for resistance to downy mildew (Plasmopara halstedii (Farl.) Berl. & de Toni) race 1. Degenerate oligonucleotide primers targeted to conserved NBS DNA sequence motifs were used to amplify RGC fragments from sunflower genomic DNA. PCR products were cloned, sequenced, and assigned to 11 groups. RFLP analyses mapped six RGC loci to three linkage groups. One of the RGCs (Ha-4W2) was linked to Pl1, a downy mildew resistance gene. A cleaved amplified polymorphic sequence (CAPS) marker was developed for Ha-4W2 using gene-specific oligonucleotide primers. Downy mildew susceptible lines (HA89 and HA372) lacked a 276-bp Tsp5091 restriction fragment that was present in downy mildew resistant lines (HA370, 335, 336, 337, 338, and 339). HA370 x HA372 F2 progeny were genotyped for the Ha-4W2 CAPS marker and phenotyped for resistance to downy mildew race 1. The CAPS marker was linked to but did not completely cosegregate with Pl1 on linkage group 8. Ha-4W2 was found to comprise a gene family with at least five members. Although genetic markers for Ha-4W2 have utility for marker-assisted selection, the RGC detected by the CAPS marker has been ruled out as a candidate gene for Pl1. Three of the RGC probes were monomorphic between HA370 and HA372 and still need to be mapped and screened for linkage to disease resistance loci.     

Analysis of total DNA by minisatellite and simple-sequence repeat primers for the use of population studies in Plasmopara halstedii

Total DNA of Plasmopara halstedii isolates from Germany was analysed for polymorphisms potentially useful for the differentiation of field isolates with respect to epidemiological studies or pathotype characterization. The isolation of the DNA started from mitotically formed zoosporangia, which is the only cellular structure of the biotrophic pathogen accessible independently from its host. The total DNA of the pathogen was used to perform DNA fingerprints with minisatellite and simple-sequence repeat primers. Polymorphisms were found that allowed differentiation on the level of single field isolates; however, they were not correlated with either physiological races or the geographic origin of the isolates. Using such differentiating primers, single spore strains of three pathogen isolates were also analysed with respect to genetic homogeneity. Minor variation was visible in the mitotically derived offspring, but the overall appearance of these patterns was mostly uniform with those of the respective parental isolate.     

Consensus mapping of major resistance genes and independent QTL for quantitative resistance to sunflower downy mildew

Major gene resistance to sunflower downy mildew (Plasmopara halstedii) races 304 and 314 was found to segregate independently from the resistance to races 334, 307 and 304 determined by the gene Pl2, already positioned on Linkage Group (LG) 8 of sunflower molecular maps. Using a consensus SSR-SNP map constructed from the INEDI RIL population and a new RIL population FU?×?PAZ2, the positions of Pl2 and Pl5 were confirmed and the new gene, denoted Pl21, was mapped on LG13, at 8?cM from Pl5. The two RIL populations were observed for their quantitative resistance to downy mildew in the field and both indicated the existence of a QTL on LG8 at 20-40?cM from the major resistance gene cluster. In addition, for the INEDI population, a strong QTL on LG10, reported previously, was confirmed and a third QTL was mapped on LG7. A growth chamber test methodology, significantly correlated with field results, also revealed the major QTL on LG10, explaining 65?% of variability. This QTL mapped in the same area as a gene involved in stomatal opening and root growth, which may be suggested as a possible candidate to explain the control of this character. These results indicate that it should be possible to combine major genes and other resistance mechanisms, a strategy that could help to improve durability of sunflower resistance to downy mildew.     

Molecular detection of Marteilia sydneyi, pathogen of Sydney rock oysters

The life cycle of Marteilia sydneyi, the aetiological agent of QX disease in the Sydney rock oyster Saccostrea commercialis, is not known. We have developed and optimised 2 diagnostic assays, the polymerase chain reaction (PCR) and in situ hybridisation, for use in investigating the role of possible alternative hosts in the life cycle of this pathogen. PCR primers, designed within the ITS1 rDNA of M. sydneyi, amplified a 195 bp fragment. Sensitivity of the PCR assay was assessed using DNA extracted from known numbers of sporonts purified from infected oyster digestive gland. DNA equivalent to 0.01 sporonts was detectable following agarose gel electrophoresis. The potential inhibitory effect of the presence of host DNA on the PCR assay was tested by the addition of oyster genomic DNA during amplification. Concentrations of host DNA in excess of 50 ng per 20 microliters reaction reduced the sensitivity of the test. Environmental validation of the PCR assay was demonstrated by the amplification of M. sydneyi DNA from 50 ng of genomic DNA extracted from QX-infected oysters. A DNA probe was constructed using the M. sydneyi unique primers and was able to detect 10 pg of M. sydneyi PCR amplified DNA in dot-blot hybridisations. The probe hybridised with presporulating and sporulating M. sydneyi stages in paraffin sections of oyster digestive gland. No non-specific binding was observed. Hybridisation consistency and signal intensity decreased as sporonts matured. While the high sensitivity and specificity of the PCR test will allow rapid screening of large numbers of potential alternative hosts for the presence of parasite DNA, it does not actually identify infective stages. In situ hybridisation conducted on paraffin sections will determine the location of the parasite within the host for morphological characterisation.     

Loop-mediated isothermal amplification assay for the detection of Plasmopara viticola infecting grapes

Grapes downy mildew caused by obligate oomycete plant pathogen Plasmopara viticola is a devastating disease worldwide, resulting in significant yield and quality losses. A field survey was conducted in two major grapes cultivated areas of Tamil Nadu for the incidence of grapevine downy mildew. The disease incidence was 43.42%–76.69%, and the highest disease incidence of 76.69% was observed in the Theni district. Totally eight P. viticola isolates were collected from different places in Coimbatore and Theni districts. These isolates were confirmed through microscopic observation and sequencing of COX 2 gene, and the phylogenetic tree was developed to study their phylogenetic relationship among the isolates which shows 97–100% sequence similarity with other P. viticola isolates and less sequence similarity with Plasmopara species. The loop-mediated isothermal amplification (LAMP) assay was developed based on the CesA4 gene sequence of P. viticola. The assay developed was more sensitive as it detected P. viticola genomic DNA up to 20 fmg. LAMP assay specificity was proved by carrying out the assay with genomic DNA extracted from other Oomycetes and fungal plant pathogens. Finally, LAMP assay was validated by testing seventy-eight grapevine leaf samples collected from seven different locations. LAMP assay showed a positive reaction in sixty-two samples tested out of seventy-eight samples tested. Therefore, the LAMP assay described should helpful for early and specific detection of downy mildew pathogen and help in mitigating disease incidence.     

Molecular analysis of a major locus for resistance to downy mildew in sunflower with specific PCR-based markers

Resistance of sunflower to the obligate parasite Plasmopara halstedii is conferred by specific dominant genes, denoted Pl. The Pl6 locus confers resistance to all races of P. halstedii except one, and must contain at least 11 tightly linked genes each giving resistance to different downy mildew races. Specific primers were designed and used to amplify 13 markers covering a genetic distance of about 3 cM centred on the Pl6 locus. Cloning and sequence analysis of these 13 markers indicate that Pl6 contains conserved genes belonging to the TIR-NBS-LRR class of plant resistance genes. Received: 9 April 2001 / Accepted: 10 August 2001